The Hox and the Distalless (Dlx) gene complexes play a major role in establishing pattern formation over the anterior/posterior axis (A/P) of the body and over the proximal/distal (P/D) axis of the limbs in mammals. The Hox genes regulate A/P axis patterning from the tail to the hind brain/midbrain junction, while the Dlx genes regulate patterning from the hindbrain to the forebrain. The Hox and Dlx clusters are linked chromosomally with a one megabase interval separating the two clusters. The expression of the Hox and Dlx genes is regulated by transcription factors and morphogens such as retinoic acid that bind enhancers and other protein binding sites in non-coding domains within the gene clusters. The protein binding sites have been shown to exert important cis regulatory control with respect to the orderly activation and display of Hox and Dlx genes over the A/P body and (P/D) limb axes. Little is known about the number, location, composition, and function of these control domains. Our previous reports on the early enhancer (EE) of the Hoxc8 gene provide the most detailed information of one such site, but much remains to be learned even about the EE, not to mention the myriad of additional sites. In this grant application, we describe our plan to investigate in greater detail the stucture and function of Hox and Dlx cis- regulatory sites. We will concentrate on two domains with which we have had experience, namely the Hoxc8 (40 kb) and Dlx3/7 (80 kb) regions. New methods of our design will be used to locate, describe, and mutate control motifs in these domains. An important new aspect of our research is the identification and functional analysis of transcription factor proteins that bind to control motifs. We view enhancer elements as the ultimate target of developmental signaling pathways. The enhancer in its complexity can be viewed as a decoding device to integrate multiple signals and to elicit appropriate gene expression responses in competent cells. Knowledge regarding enhancer sequence, protein binding domains, and the specific transcription factors binding the enhancers will contribute to our understanding of the epigenetic function of these motifs. We believe an in depth investigation of non-coding control elements in the Hoxc8 and Dlx3/7 domains will provide insightful information relevant to the entire clusters with respect to their control of developmental patterning and their role in the evolutionary diversification of body plans.